The present invention is generally in the area of oligomers, and more specifically in the area of nucleotide sequences that block the function of DNA-dependent protein kinase.
Ku protein, a heterodimer of 70 kDa and 83 kDa polypeptides, is the regulatory component of the DNA-dependent protein kinase (DNA-PK). Ku protein binds to DNA discontinuities and is essential for DNA double-strand break repair.
Ku protein was first identified as an autoantigen in sera from certain patients with autoimmune disease (Mimori et al., J. Biol. Chem. 261, 2274-2278 (1986)). Subsequent characterization showed that Ku protein binds avidly to double-stranded DNA ends and other structural discontinuities in DNA such as nicks, gaps, and hairpins (Mimori and Hardin, J. Biol. Chem. 261, 10375-10379 (1986); Paillard and Strauss, Nucleic Acids Res. 19, 5619-5624 (1991); Zhang and Yaneva, Biochem. Biophys. Res. Commun. 186, 574-479 (1992); Blier et al., J. Biol. Chem. 268, 7594-7601 (1993); Falzon et al., J. Biol. Chem. 268, 10546-10552 (1993)). Further biochemical analysis demonstrated that Ku protein is the regulatory component of the DNA-dependent protein kinase (Dvir et al., Proc. Natl. Acad. Sci. USA 89, 11920-11924 (1992); Gottlieb and Jackson, Cell 72, 131-142 (1993)). In the presence of DNA ends, Ku protein can interact with the catalytic subunit of DNA-PK (DNA-PKcs) which is thereby targeted to the DNA. The ability of Ku protein to interact with DNA ends suggested that Ku and DNA-PKcs may play a role in DNA repair and recombination (Anderson, Trends Biochem. Sci. 18, 433-437 (1993)). Subsequent characterization of ionizing radiation-sensitive mutant cell lines showed that Ku protein and DNA-PKcs are essential for repair of DNA double-strand breaks and for V(D)J recombination (Getts and Stamato, J. Biol. Chem. 269, 15981-15984 (1994); Rathmell, Proc. Natl. Acad. Sci. USA 91, 7623-7627 (1994); Smider et al., Science 266, 288-291 (1994); Taccioli et al., Science 265, 1442-1445 (1994); Blunt et al., Cell 80, 813-823 (1995); Boubnov et al., Proc. Natl. Acad. Sci. USA 92, 890-894 (1995); Kirchgessner et al., Science 267, 1178-1183 (1995); Lees-Miller et al., Science 267, 1183-1185 (1995); Peterson et al., Proc. Natl. Acad. Sci. USA 92, 3171-3174 (1995)).
The binding of Ku protein to double-stranded DNA ends is largely sequence-independent. The ability of Ku protein to undergo facilitated transfer between DNA fragments with cohesive ends suggests that Ku protein may be able to interact transiently with two DNAs simultaneously, perhaps serving to align the ends for ligation (Bliss and Lane, J. Biol. Chem. 272, 5765-5773 (1997)). Consistent with this, recent atomic force microscopy and electron microscopy studies show images of Ku protein tethering DNA fragments together and participating in loop structures (Cary, Proc. Natl. Acad. Sci. USA 94, 4267-4272 (1997); Pang et al., Cancer Res. 57, 1412-1415 (1997)). There have also been a number of reports of possible sequence-specific binding of Ku protein to DNA (for example, Knuth et al., J. Biol. Chem. 265, 17911-17920 (1990); Messier et al., Proc. Natl. Acad. Sci. USA 90, 2685-2689 (1993); Okumura et al., FEBS Lett. 356, 94-100 (1994); Roberts et al., Proc. Natl. Acad. Sci., USA 91, 6354-6358 (1994)). Most recently, a sequence in the long terminal repeat of mouse mammary tumor virus has been characterized that appears to allow interaction of Ku protein with DNA in the absence of ends or single-stranded regions (Giffin et al., Nature 380, 265-268 (1996); Giffin et al., J. Biol. Chem. 272, 5647-5658 (1997)).
There is some evidence that Ku protein interacts with RNA, although this has been much less studied than the interaction with DNA. Antibodies to Ku protein stain both the nucleoplasm and the nucleolus. The amount of Ku protein in the nucleolus changes depending on the growth state of the cell, suggesting that this localization is actively regulated (Yaneva and Jhiang, Biochim. Biophys. Acta 1090, 181-187 (1991)). Separately, it has been demonstrated that nucleolar staining is sensitive to RNase treatment, whereas nucleoplasmic staining is not (Reeves, J. Exp. Med. 161, 18-39 (1985)). Thus, nucleolar localization may be regulated by interaction of Ku protein with RNA. Ku protein does not appear to bind to bulk tRNA or to synthetic RNA polymers (Mimori and Hardin, J. Biol. Chem. 261, 10375-10379 (1986)). However, one study showed that Ku protein forms a specific complex with an RNA that included the HIV trans-activation response (TAR) element sequence (Kaczmarski and Khan, Biochem. Biophys. Res. Commun. 196, 935-942 (1993)).
The Ku protein has been suggested to be involved in many important nuclear processes, including transcription, replication, and growth control, as well as DNA repair. It would be useful to have a means to demonstrate whether Ku protein is required for a biochemical activity (such as repair or recombination) that has been reconstituted in a crude cell-free system. It would also be useful to have a means to identify nuclear proteins that physically interact with Ku protein to exert their biological functions.
Ku protein and DNA-PK are important in the repair of radiation-induced DNA damage. If damage cannot be repaired, cells die. The cytotoxic effect of ionizing radiation forms the basis for radiation therapy, which is widely used in the treatment of human cancer. The efficacy of radiation therapy is currently limited by the radiation resistance of certain tumors (for example, glioblastomas) and by the side effects caused by irradiation of nearby normal tissues (for example, in treatment of breast and cervical cancer). Therefore, it would also be useful to have a means for sensitizing target cells and tissues to therapeutic radiation.
Some patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and scleroderma produce anti-Ku antibodies. Ku is one of a number of proteins that are targets of autoantibodies in these patients. High levels of autoantibodies lead to deleterious consequences for the patient. It would be useful to have a compound directed against Ku protein to alter the course of autoimmune disease in patients with anti-Ku antibodies.
A problem in gene therapy is a loss of the foreign DNA from illegitimate recombination. It is believed that illegitimate recombination requires Ku-dependent double strand break repair. It would be useful to have a means to improve the stability of transgene DNA.
Therefore, it is an object of the disclosed invention to provide oligomers that bind to Ku protein.
It is also an object of the disclosed invention to provide oligomers that prevent illegitimate recombination to improve the stability of transgene DNA.
It is another object of the disclosed invention to provide oligomers that recognize Ku protein in a complex environment containing other macromolecules.
It is also an object of the disclosed invention to provide oligomers that improve the efficacy of radiation therapy by inhibiting DNA repair in the target cells.
It is also an object of the disclosed invention to provide oligomers to treat autoimmune disease in patients with anti-Ku antibodies.
It is also an object of the disclosed invention is to provide an assay for cellular proteins that interact with Ku protein or that promote or inhibit the interaction between Ku protein and DNA-PKcs.
It is also an object of the disclosed invention is to provide oligomers useful for manipulating the activity of Ku protein in cells and organisms to better understand its physiological role.
Disclosed are oligomers that bind Ku protein. These oligomers, also referred to herein as aptamers, are useful for inhibiting activation of DNA-PK, treating certain forms of autoimmune disease, detection and purification of Ku protein, and identification of proteins that interact with Ku protein. Preferably, the oligomers are composed of nucleotides, nucleotide analogs, or a combination. Most preferably, the oligomers are composed of ribonucleotides. Also disclosed is a method of inhibiting DNA repair, a method of identifying cellular proteins that interact with Ku protein, and a method of treating autoimmune disease in patients with anti-Ku antibodies.
The disclosed oligomers can have several preferred features, either alone or in combination, in addition to Ku binding. One such feature, referred to herein as inhibition activity, is inhibition of DNA-PK kinase activity. As discussed above, interaction of the Ku protein and DNA is involved in activation of DNA-PK kinase activity. Another preferred feature, referred to herein as aptamer motifs, is the presence of one or more of the base sequences GCUUUCCCANNNAC, (SEQ ID NO:20) A(A/C)AUGA, (SEQ ID NO:21) and AACUUCGA. These sequencesxe2x80x94referred to herein as aptamer motif 1, aptamer motif 2, and aptamer motif 3, respectivelyxe2x80x94are associated with Ku binding capability. Another preferred feature, referred to herein as aptamer structure, is the presence of a structure similar to the structure shown in FIG. 6A. This structure has the general formula 5xe2x80x2-A-B-C-D-Cxe2x80x2-E-Axe2x80x2-3xe2x80x2, where A, B, C, D, Cxe2x80x2, E, and Axe2x80x2 are components of the oligomer. In this structure, A and Axe2x80x2 interact to form a stem structure, C and Cxe2x80x2 interact to form a stem structure, B and E make up a bulge region, and D is either a bulge or a loop. FIG. 6A depicts component D as a loop. Each of these preferred features (inhibition activity, aptamer motif, and aptamer structure) can be used either alone or in combination with one or both of the other characteristics.